Peptide substrates for the identification of factor Xa

ABSTRACT

Peptide substrates selected from the group consisting of (i) the tri- and tetra-peptides of the formula 
     
         Q-A.sub.1 -A.sub.2 -Gly-A.sub.4 -R                         (I) 
    
     having SEQ ID NO: 1 in which Q is a group which blocks the N-termnal end of the peptide chain, A 1  is selected from the group consisting of Leu, Ile, Nle, Nva, CHA, CHG, CHT, Phe, Ala and Lys groups, where the basic side-group of the Lys residue is blocked with a suitable group which substantially removes the basic character of said side-group, A 2  is a single bond, Asp or Glu, the carboxylic acid side-group of Asp and Glu being capable of esterification or amidation, A 4  is selected from the group consisting of Arg and Lys groups, and R is pNA; and (ii) their addition salts. These peptide substrates are useful in the determination of Factor Xa.

FIELD OF THE INVENTION

The present invention relates, by way of novel industrial products, totri- and tetra-peptide compounds. These novel products are particularlyuseful as substrates for the identification and assay of Factor Xa (i.e.activated Factor X) involved in the mechanisms of hemostasis. Theinvention further relates to the method of preparing these novelproducts and to the method of assaying and/or identifying Factor Xa bymeans of said products.

PRIOR ART

The enzymes belonging to the class E.C. 3.4.21 [as defined in the work"Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam1973, pages 238 et seq. (former nomenclature: class E.C. 3.4.4)] areknown to be substances which cleave the amide linkages of the protein orpeptide backbone at the carboxyl group of Arg, Lys, Orn and Hisresidues. This cleavage mechanism is well known to those skilled in theart and is amply exemplified in the documents of the prior art citedbelow.

The currently used substrates for enzymes belonging to said class areessentially tri- or tetra-peptide compounds whose N-terminal end isgenerally substituted by a blocking group such as benzoyl,benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, tosyl, acetyl orthe like and whose CO-terminal end is amidated by an aminated groupwhich can be a radioactive radical or a radical, especially ap-nitroanilino group, capable of imparting coloration or fluorescencebefore or (preferably) after cleavage. Reference is made in thisconnection to the following patent documents: FR-A-2 372 798, EP-A-0 004256, U.S. Pat. Nos. 4,508,644, 4,448,715, FR-A-2 471 411, FR-A-2 317 280and FR-A-2 459 226.

It is found that the peptide derivatives in the patent documents citedabove have little affinity for water. They have a low solubility ordispersibility in water; consequently, it is sometimes necessary to addan organic solvent to make them usable. The systems comprising mixedsolvents are scarcely compatible, in general terms, with biologicalmedia: they cause either a decrease in the activity of the substrate or,in certain cases, a deterioration in the enzyme which it is desired toassay. Furthermore, the low affinity of these substrates for watercauses a decrease in the sensitivity of the enzyme assay methods.

To improve the solubility of enzyme substrates in water, EP-A-0 025 190has disclosed a first technical solution which consists in substituting,on said N-terminal end, a polyethylene glycol residue monoetherified byan alkyl group, for example Me--O(CH₂ CH₂ O)_(x) --CO (in which X is aninteger such that the polyethylene glycol residue has an averagemolecular weight of about 600), the amino acid residue containing theCO-terminal end of the peptide chain being other than the Arg and Lysresidues which are present according to the invention, as will be seenbelow; also, EP-A-0 280 610 (to the Applicant) has disclosed anothertechnical solution which consists in attaching analkoxycarbonylmethylenecarbonyl radical, such as the methoxymalonylradical (i.e. Me--O--CO--CH₂ --CO, abbreviated to MM), to the N-terminalend of a dipeptide.

Furthermore, according to document EP-A-0 280 610 cited above, dipeptidesubstrates containing said methoxymalonyl radical on said N-terminal endare generally presented as being more sensitive towards enzymesbelonging to the class E.C. 3.4.21 than tri- and tetra-peptidesubstrates containing said methoxymalonyl radical. However, according toEP-A-0 280 610, these dipeptide substrates are not particularly specifictowards Factor Xa and do not permit an effective assay of said FactorXa.

Factor Xa, which belongs to the subclass E.C. 3.4.21.6, is known to beone of the important components involved in the mechanisms ofhemostasis. The activation of Factor X leads to the formation of FactorXa, which is a serine protease responsible for the conversion ofprothrombin to thrombin. It is therefore of very great interest, fromthe diagnostic point of view, to have peptide substrates which arespecific for Factor Xa and which can be used by a simple determinationtechnique before any adverse thromboembolic accident.

It is known that tri- and tetra-peptide substrates have already beenrecommended for the determination of Factor Xa. The following are knownin particular among the tripeptide substrates which have been proposedfor this purpose:

from EP-A-0 004 256 cited above, ##STR1## and their acid addition salts;from EP-A-0 110 306,

    Z-D-Leu-Gly-L-Arg-pNA

(presented as illustrating the prior art; see Table VIII on page 32 ofsaid document EP-A-0 110 306),

    Z-D-Leu-Gly-L-Arg(2-OMe)pNA,

    Z-D-Leu-Gly-L-Arg(2-CONHMe)pNA,

    Z-D-Leu-Gly-L-Arg(2-COOBu)pNA,

    H-D-Lys(Z)-Gly-L-Arg(2-Z)pNA,

    Tos-Gly-L-Pro-L-Arg(2-CONHiPr)pNA

and their addition salts; and

from U.S. Pat. Nos. 4,440,678 and 4,480,030 (which correspond to EP-A-0034 122), the compounds of the formula ##STR2## in which X¹ is C₂ -C₈alkanoyl, C₂ -C₈ ω-aminoalkanoyl, phenylalkanoyl (in which the alkanoylfragment is C₂ -C₄ and the phenyl fragment can be substituted in thepara position by NH₂), cyclohexylcarbonyl (which can be substituted inthe 4 position by MeNH), benzoyl (which can be substituted in the orthoor para position), alkoxycarbonyl (in which the alkoxy fragment is C₁-C₈), benzyloxycarbonyl (in which the benzyloxy fragment can besubstituted in the para position by MeO, Me or Cl), C₁ -C₄alkanesulfonyl, phenylsulfonyl (in which the phenyl fragment can besubstituted in the para position by Me) or α- or β-naphthylsulfonyl,

X² is C₁ -C₆ alkyl with a linear or branched hydrocarbon chain, C₁ -C₂hydroxyalkyl, alkoxyalkyl (in which the alkoxy fragment is C₁ -C₄ andthe alkyl fragment is C₁ -C₂), ω-carboxyalkyl or ω-alkoxycarbonylalkyl(in which the alkyl fragments are C₁ -C₃ and the alkoxy fragment is C₁-C₄), ω-benzyloxycarbonylalkyl (in which the alkyl fragment is C₁ -C₃),cyclohexyl, cyclohexylmethyl, 4-hydroxycyclohexylmethyl, phenyl, benzyl,4-hydroxybenzyl or imidazol-4-ylmethyl, with the proviso that X¹ isother than the benzyloxycarbonyl group when X² is isopropyl orcyclohexyl,

X³ is C₁ -C₄ alkyl,

X⁴ is H, Me or Et and

X⁵ is an aminated group containing an amino group substituted by anaromatic or heterocyclic radical, which can be cleaved by enzymichydrolysis to give a colored or fluorescent compound H--X⁵ detectable byphotometry, spectrophotometry or fluorophotometry, the amount of H--X⁵released being proportional to the amount of enzyme causing the cleavageof the peptide substrate,

and their acid addition salts.

The following tripeptides: ##STR3## and their acid addition salts, maybe mentioned in particular among the compounds of formula Io which havebeen proposed for the determination of Factor Xa.

The following are known in particular among the tetrapeptide compoundswhich have been described as substrates useful in the determination ofFactor Xa:

the compound S-2222 (marketed by Kabi Diagnostica, Stockholm, Sweden),which has the formula

    Bz-L-Ile-Glu-Gly-L-Arg-pNA.HCl;

from CH-A-637 627 (corresponding to FR-A-2 372 798 cited above), thecompounds ##STR4## and their acid addition salts; and from EP-A-0 010306 cited above,

    Bz-L-Ile-L-Glu(OMe)-Gly-L-Arg-(2-CONHMe)pNA

and its acid addition salts.

The prior art relating to the tri- or tetra-peptide substratesrecommended for the determination of Factor Xa teaches that thespecificity is a function of (i) the nature of the peptide chain, (ii)the structure of the blocking group at the N-terminal end, and (iii) thechoice of cleavable developing group at the CO-terminal end of saidchain.

SUBJECT OF THE INVENTION

According to the invention, a novel technical solution is recommendedwhich differs from the prior art in the choice of a group which (i)blocks the N-terminal end of the peptide chain, and (ii) improves thesolubility in water or the affinity for water of tri- or tetra-peptidecompounds useful in the determination of Factor Xa.

Thus, according to a first feature of the invention, peptide compoundsare recommended as novel industrial products, said compounds beingselected from the group consisting of (i) the tri- and tetra-peptides ofthe formula, the tetra-peptide being identified as SEQ ID NO:1,

    Q-A.sub.1 -A.sub.2 -Gly-A.sub.4 -R                         (I)

in which

Q is a group which blocks the N-terminal end of the peptide chain andwhich is selected from the group consisting of

(a) an oxymalonyl radical of the formula R₁ --O--CO--CH₂ --CO, in whichR₁ is

a C₁ -C₄ alkyl group,

a phenyl group,

a phenyl group substituted by one or more Me, MeO, Cl, Br, F or CF₃groups,

a C₃ -C₆ cycloalkyl group,

a benzyl group,

a benzyl group substituted by one or more Me, MeO, Cl, Br, F or CF₃groups, or

a cycloalkylmethyl group in which the cycloalkyl fragment is C₃ -C₆ ;and

(b) a polyethylene glycol residue of the formula R₂ --

O(CH₂ CH₂ O)_(n) --CO, in which R₂ is a C₁ -C₆ alkyl, phenyl or benzylgroup and n is an integer having a value of 1 to 170,

A₁ is selected from the group consisting of Leu, Ile, Nle, Nva, CHA,CHG, CHT, Phe, Ala and Lys groups, where the basic side-group of the Lysresidue is blocked with a suitable group which substantially removes thebasic character of said side-group,

A₂ is a single bond, Asp or Glu, the carboxylic acid side-group of Aspand Glu being capable of esterification or amidation,

A₄ is selected from the group consisting of Arg and Lys groups, and

R is a cleavable labeling means; and

(ii) their addition salts.

According to a second feature of the invention, it is recommended to usethe compounds of formula I and their acid addition salts as enzymesubstrates for the determination of Factor Xa.

It has in fact been found, surprisingly, that the tri- andtetra-peptides according to the invention are more valuable than thetri- and tetra-peptide substrates known in the prior art in the field ofthe determination of Factor Xa, either because they are more active, orbecause they are more selective, or else because they are morewater-soluble than said substrates of the prior art.

According to another feature of the invention, a method of preparing thecompounds of formula I and their addition salts is recommended.

According to yet another feature of the invention, a method ofdetermining Factor Xa by means of the compounds of formula I or theiraddition salts is provided.

Abbreviations

For convenience, the following abbreviations have been used in thepresent description:

the amino acid residues:

Ala=α-alanyl

β-Ala=β-alanyl

AOA=α-aminooctanoyl

Arg=arginyl Asp=α-aspartyl

CHA=3-cyclohexylalanyl

CHG=α-cyclohexylglycyl

CHT=3-(4-hydroxycyclohexyl)alanyl

Ile=isoleucyl

Leu=leucyl

Lys=lysyl

MeGly=N-methylglycyl (or sarcosyl)

Nle=norleucyl

Nva=norvalyl

Phe=phenylalanyl

Pro=prolyl

Pyr=pyroglutaminyl (or pyrrolidin-2-one-5-carbonyl)

the other abbreviations:

Ac=acetyl

AcOH=acetic acid

Adoc=adamantyloxycarbonyl

Aoc=t-amyloxycarbonyl

Boc=t-butoxycarbonyl

Bop=(benzotriazol-1-yl)oxytris(dimethylamino)phosphoniumhexafluorophosphate (alternative nomenclature: CASTRO's reagent) of theformula ##STR5## Bu=n-butyl Bz=benzoyl

Bzl=benzyl

Cbo=carbobenzoxy

DCCI=dicyclohexylcarbodiimide

DIEA=diisopropylethylamine

DMF=dimethylformamide

Et=ethyl

EM=ethoxymalonyl (EtO--CO--CH₂ --CO)

Et₃ N=triethylamine

EtO=ethoxy

Fmoc=fluoren-9-ylmethoxycarbonyl

Foc=furfuryloxycarbonyl

HMPT=N,N,N',N',N",N"-hexamethylphosphorotriamide

HOBT=1-hydroxybenzotriazole

HPLC=high performance liquid chromatography

H-TFA=trifluoroacetic acid (or HTFA)

Iboc=isobornyloxycarbonyl

iBu=isobutyl

iPr=isopropyl

Me=methyl

MeO=methoxy

MeOH=methanol

MM=methoxymalonyl (MeO--CO--CH₂ --CO)

Mor=morpholin-1-yl

MW=molecular weight

OD=optical density

OSu=N-oxysuccinimide or (2,4-dioxopyrrolidin-1-yl)oxy

PEG=polyethylene glycol

Ph=phenyl

pH=cologarithm of the concentration of H⁺ ions

Pi=piperidin-1-yl

pNA=p-nitroanilino or (4-NO₂)C₆ H₄ NH

Pr=n-propyl

Py=pyrrolidin-1-yl

RT=room temperature (15-20° C.)

tBu=t-butyl

THF=tetrahydrofuran

TLC=thin layer chromatography

Tos=p-toluenesulfonyl (or tosyl)

TR=retention time

Z=benzyloxycarbonyl

Z(p-Cl)=p-chlorobenzyloxycarbonyl

Z(p-OMe)=p-methoxybenzyloxycarbonyl

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the group Q is a group which blocks the N-terminalend of the peptide chain A₁ -A₂ -Gly-A₄, identified as SEQ ID NO: 1(when residue A₁ is an amino acid in the L-configuration and A₂ is anAsp or Glu residue). It is an oxymalonyl radical of the formula R₁--O--CO--CH₂ --CO or a PEG residue of the formula R₂ --O(CH₂ CH₂ O)_(n)--CO. In said oxymalonyl radical, R₁ can be especially

a C₁ -C₄ alkyl group, preferably Me, Et, Pr, iPr, Bu or tBu,

a phenyl group,

a tolyl, xylyl, 2-, 3- or 4-methoxyphenyl, 2,4-, 2,6-, 3,4- or3,5-dimethoxyphenyl, 4-chlorophenyl, 4-fluorophenyl, 3,4-, 3,5- or2,6-dichlorophenyl or 3-trifluoromethylphenyl group,

a cyclopropyl, cyclopentyl or cyclohexyl group,

a benzyl group,

a 2-, 3- or 4-methylbenzyl, 2-, 3- or 4-methoxybenzyl, 2,4-, 2,6-, 3,4-or 3,5-dimethylbenzyl, 2,4-, 2,6-, 3,4- or 3,5-dimethoxybenzyl, 2-, 3-or 4-chlorobenzyl, 2-, 3- or 4-fluorobenzyl, 2,4-, 2,6-, 3,4- or3,5-dichlorobenzyl or 3-trifluoromethylbenzyl group, or

a cyclopropylmethyl, cyclopentylmethyl or cyclohexylmethyl group.

When R₁ is a substituted phenyl or substituted benzyl group, thesubstituents will advantageously be located in the 2, 3 and/or 4position. Even more advantageously, R₁ will be Me or Et so as to givethe peptide compound a greater affinity for water; in other words, Qwill then be MM or EM.

As regards the fragment O(CH₂ CH₂ O)_(n) of the PEG residue, n will bean integer having a value of 1 to 170, as indicated above. Inparticular, the average MW of the fragment O(CH₂ CH₂ O)_(n) will bebetween about 60 (n=1) and about 7100 (n=161). In said PEG residue, R₂will advantageously be Me, Et, Pr, iPr, Bu, tBu or n-hexyl; even moreadvantageously, R₂ will be Me or Et.

As indicated above, A₁ is an amino acid residue selected from the groupconsisting of Leu, Ile, Nle, Nva, CHA, CHG, CHT, Phe, Ala and Lys, wherethe basic side-group of the Lys residue is blocked with a suitable acidgroup [such as Boc, Cbo, Z, Z(p-Cl), Z(p-OMe), Adoc, Aoc, Fmoc, Foc,Iboc]. In the case of the Lys residue, such an acid group makes itpossible to remove the basic character of the basic side-group forpractical purposes.

The residue A₁ can have the L or D configuration; however, for a betteractivity or sensitivity as regards the determination of Factor Xa, saidresidue A₁ preferably has the D configuration.

It is for this reason that A₁ will advantageously be selected from thegroup consisting of D-Leu, D-Ile, D-Nle, D-Nva, D-CHA, D-CHG, D-CHT,D-Phe, D-Ala and D-Lys groups, where the basic side-group of D-Lys isblocked with a suitable acid group.

The residues A₁ which are considered to be the most valuable accordingto the invention are D-Nle, D-Leu and D-CHA.

As indicated above, A₂ is a single bond or an Asp or Glu residue, itbeing possible, if appropriate, for the carboxyl side-group of each Aspand Glu residue to be esterified [i.e. for the OH group of the COOHgroup to be replaced with a group OR₃, in which R₃ is especially C₁ -C₄alkyl (for example Me, Et, iPr, Pr, Bu, tBu or iBu), C₁ -C₄ hydroxyalkyl(preferably CH₂ CH₂ --OH), C₃ -C₆ cycloalkyl (such as cyclopropyl,cyclopentyl or, preferably, cyclohexyl) or ω-aminoalkyl, in which thealkyl fragment is C₂ -C₄ and in which the nitrogen atom of the aminofragment can be monoalkylated (NHR₄), dialkylated (NR₄ R₅) or includedin a ring (NR₄ R₅ =N-heterocyclic group)] or amidated [i.e. for the OHgroup of the COOH group to be replaced with a group NH₂, NHR₄ or NR₄ R₅(in which R₄ and R₅, which are identical or different, are each a C₁ -C₄alkyl group, it being possible for R₄ and R₅, taken together, to form,with the nitrogen atom to which they are bonded, a 5- to 7-memberedN-heterocyclic group selected in particular from the group consisting ofpyrrolidino, piperidino, morpholino, thiomorpholino, piperazino,4-(2-hydroxyethyl)piperazino, 4-methylpiperazino,4-(4-chlorophenyl)piperazino and hexamethyleneimino groups)].

Examples of Glu and Asp residues in which the COOH side-group isesterified or amidated are given in documents CH-A-637 627 and FR-A-2372 798 cited above, which are incorporated here by way of reference.

Advantageously, when A₂ is an amino acid residue, said residue will havethe L configuration; A₂ will therefore be either a single bond or a Gluor Asp residue selected especially from the group consisting of L-Glu,L-Asp, L-Glu(Mor), L-Glu(Py), L-Glu(Pi), L-Glu(OMe), L-Glu(OEt),L-Glu(OtBu), L-Asp(Mor), L-Asp-(Py), L-Asp(Pi), L-Asp(OMe), L-Asp(OEt)and L-Asp(OtBu) groups.

A₄ will also advantageously be L-Arg or L-Lys.

The labeling means R is well known in the art of biological andmicrobiological assays; reference is made in this connection to theprior art cited above and especially document U.S. Pat. No. 4,448,715.Said labeling means will preferably be selected from the group ofaminated groups NH--R' which (i) induce a color change, (ii) induce achange in fluorescence, or (iii) contain at least one radioactiveelement (for example an anilino or benzylamino group labeled with a ¹⁴ Cor ³ H radioisotope). Any amino group NH--R' which gives, during orafter the enzymic reaction, a signal capable of being amplified fordetection (for example by measurement of the optical density at a givenwavelength, or by measurement of the radioactivity) is suitable for thepurposes of the invention. The amount of product H--R obtained bycleavage in the enzymic hydrolysis is proportional to the amount ofenzyme used. Said amount of H--R can be determined by photometry,spectrophotometry, fluorospectrophotometry or electrochemistry.

The group R which is preferred according to the invention is achromogenic group, typically a nitrophenylamino group (in which thephenyl radical is capable of being substituted by a group COOH, F, Cl,Br, CH₃, OCH₃, CN, CF₃ and/or SO₃ H), or a fluorogenic group, typicallya naphthylamino group (in which the naphthyl radical is capable of beingsubstituted by a group OCH₃, COOH, SO₃ H or CH₃), and4-methylcoumaryl-7-amino, 4-trifluoromethylcoumaryl-7-amino andanalogous groups.

The following may be mentioned in particular among the chromogenic andfluorogenic aminated groups which are suitable according to theinvention: p-nitroanilino (abbreviated to pNA), 2-carboxy-4-nitroanilinoand 3-carboxy-4-nitroanilino, 2-halogeno-4-nitroanilino and3-halogeno-4-nitroanilino (in which the halogen is F, Cl or Br),2-methoxy-5-methyl-4-nitroanilino, 2-hydroxysulfonyl-4-nitroanilino,4-trifluoromethyl-2-nitroanilino, 4-trifluoromethyl-3-nitroanilino,4-cyano-2-nitroanilino, naphthyl-2-amino,4-hydroxysulfonylnaphthyl-1-amino, quinolylamino, nitroquinolylamino andthe like.

The preferred group R according to the invention is a chromogenic group,namely on the one hand pNA and on the other hand analogous groups inwhich the phenyl ring of pNA is substituted in the 2 or 3 position, saidgroups having the formula ##STR6## in which Y is Br, Cl, F, CF₃, COOH,COOW, CONH₂, CONHW, CONW₂, CONH(CH₂)_(m) NMe₂, OH or OW, in which W is aC₃ -C₆ alkyl, C₆ -C₁₀ aryl, C₇ -C₁₁ aralkyl or C₃ -C₈ alicyclic groupand m is an integer having a value of 1 to 10.

Such groups of formula II in which the phenyl ring of the pNA group issubstituted are described especially in document EP-A-0 110 306 citedabove.

The addition salts according to the invention are essentially acidaddition salts obtained by reacting a compound of formula I with amineral or organic acid.

The best mode of carrying out the invention consists in using, as thesubstrate, a tri- or tetra-peptide compound of the formula

    Q-A.sub.1 -A.sub.2 -Gly-A.sub.4 -pNA                       (III)

in which

Q is MM, EM or R₂ --O(CH₂ CH₂ O)_(n) 13 CO, in which R₂ is Me, Et, Pr,iPr, Bu, iBu or n-hexyl and in which n is an integer of between 1 andabout 161,

A₁ is selected from the group consisting of D-Leu, D-Ile, D-Nle, D-Nva,D-CHA, D-CHG, D-CHT, D-Phe, D-Ala, D-Lys(Cbo) and D-Lys(Boc), thepreferred groups A₁ being D-Nle, D-Leu and D-CHA,

A₂ is selected from the group consisting of a single bond and L-Glu andL-Asp groups, in which the OH group of the carboxylic acid side-radicalof said L-Glu and L-Asp can be replaced with OMe, OEt, OtBu, morpholino,piperidino or pyrrolidino, and

A₄ is selected from the group consisting of L-Arg and L-Lys,

and its acid addition salts.

Without implying a limitation, a number of peptide compounds (compounds"Ex.") according to the invention have been collated in Tables I and IIbelow. For convenience, a reference peptide, which has been marketed asa substrate specific for Factor Xa, has also been included in Table I byway of comparison.

The peptide compounds according to the invention which have proved to beof particular value are especially the following:

(a) MM-D-Leu-Gly-L-Arg-pNA,

(b) EM-D-Leu-Gly-L-Arg-pNA,

(c) MeO(CH₂ CH₂ O)-CO-D-Leu-Gly-L-Arg-pNA,

(d) MeO(CH₂ CH₂ O)₂ -CO-D-Leu-Gly-L-Arg-pNA,

(e) MeO(CH₂ CH₂ O)₃ -CO-D-Leu-Gly-L-Arg-pNA,

(f) MeO(CH₂ CH₂ O)₇ -CO-D-Leu-Gly-L-Arg-pNA,

(g) MM-D-Nle-Gly-L-Arg-pNA,

(h) MM-D-Nle-L-Glu(Py)-Gly-L-Arg-pNA,

(i) MM-D-CHA-Gly-L-Arg-pNA,

(j) MM-D-Ile-L-Glu(OMe)-Gly-L-Arg-pNA,

(k) EM-D-Nle-Gly-L-Arg-pNA and

(l) their acid addition salts.

                  TABLE I                                                         ______________________________________                                        Q--A.sub.1 --A.sub.2 -Gly-A.sub.4 --pNA.HA                                      Product Q        A.sub.1  A.sub.2 A.sub.4                                                                              HA                                 ______________________________________                                        Ex. 1 MM       D-Leu      --      L-Arg  AcOH                                   Ex. 2 EM D-Leu -- L-Arg AcOH                                                  Ex. 3 MM D-CHA -- L-Arg AcOH                                                  Ex. 4 MM D-Nle -- L-Lys AcOH                                                  Ex. 5 MM D-CHT -- L-Arg AcOH                                                  Ex. 6 MM D-Nva -- L-Arg AcOH                                                  Ex. 7 MM D-CHG -- L-Arg HCl                                                   Ex. 8 MM D-Phe -- L-Arg HCl                                                   Ex. 9 MM D-Ala -- L-Arg HCl                                                   Ex. 10 MM D-Ile -- L-Arg AcOH                                                 Ex. 11 MM D-Lys(Cbo) -- L-Arg AcOH                                            Ex. 12 MM D-CHG L-Asp(Py) L-Lys HCl                                           Ex. 13 MM D-CHT -- L-Arg H-TFA                                                Ex. 14 MM D-Nle -- L-Arg HCl                                                  Ex. 15 EM D-CHA -- L-Arg HCl                                                  Ex. 16 EM D-CHT -- L-Arg HCl                                                  Ex. 17 EM D-Nva -- L-Arg AcOH                                                 Ex. 18 EM D-CHG -- L-Arg AcOH                                                 Ex. 19 EM D-Ala -- L-Arg AcOH                                                 Ex. 20 EM D-Ile -- L-Arg HCl                                                  Ex. 21 EM D-Nle -- L-Arg HCl                                                  Ex. 22 MM D-Ile -- L-Lys AcOH                                                 Ex. 23 MM D-CHG -- L-Lys AcOH                                                 Ex. 24 MM D-Nva -- L-Lys HCl                                                  Ex. 25 MM D-Lys(Boc) -- L-Arg AcOH                                            Ex. 26 MM D-Ala -- L-Arg HCl                                                  Ex. 27 MM D-Nle L-Glu(Py) L-Arg HCl                                           Ex. 28 MM D-Nle L-Glu(OMe) L-Arg AcOH                                         Ex. 29 MM D-Nle L-Asp(Py) L-Arg AcOH                                          Ex. 30 MM D-Nle L-Glu L-Arg HCl                                               Ex. 31 MM D-Leu L-Glu(OMe) L-Arg AcOH                                         Ex. 32 MM D-Ile L-Glu L-Arg AcOH                                              Ex. 33 MM D-Ile L-Glu(OMe) L-Arg AcOH                                         Ex. 34 MM D-Nva L-Glu(Mor) L-Arg AcOH                                         Ex. 35 MM D-CHT L-Asp(OMe) L-Arg AcOH                                         Ex. 36 MM D-Leu L-Asp L-Lys AcOH                                              Ex. 37 MM D-Lys(Cbo) L-Glu(Pi) L-Arg HCl                                      Ex. 38 MM D-Lys(Cbo) L-Asp(Pi) L-Arg HCl                                      Ex. 39 MM D-CHA L-Glu L-Arg AcOH                                              Ex. 40 MM D-CHG L-Glu(Py) L-Arg AcOH                                          Ex. 41 MM D-Ala L-Glu(OtBu) L-Arg AcOH                                        Ex. 42 MM D-Ala L-Asp L-Arg AcOH                                              Ex. 43 MM D-Leu L-Glu(Py) L-Arg HCl                                           Ex. 44 MM D-Leu L-Asp(OMe) L-Arg HCl                                          Ex. 45 MM D-Ile L-Asp(Py) L-Arg AcOH                                          Ex. 46 MM D-Phe L-Glu L-Arg AcOH                                              Ex. 47 MM D-Phe L-Glu(OMe) L-Arg AcOH                                         Ex. 48 MM D-Phe L-Asp L-Arg HCl                                               Ex. 49 MM D-Phe L-Asp(Pi) L-Arg HCl                                           Ex. 50 MM D-Nle L-Glu(Mor) L-Lys AcOH                                         Ex. 51 MM D-Nle L-Asp L-Arg AcOH                                              Ex. 52 MM D-Lys(Cbo) L-Glu L-Arg AcOH                                         Ex. 53 MM D-Lys(Cbo) L-Glu(OMe) L-Arg AcOH                                    Ex. 54 MM D-Lys(Cbo) L-Glu(Py) L-Arg AcOH                                     Ex. 55 MM D-Lys(Cbo) L-Asp L-Arg AcOH                                         Ex. 56 MM D-Lys(Cbo) L-Asp(Py) L-Arg AcOH                                     Ex. 57 MM D-Nva L-Glu(OMe) L-Arg AcOH                                         Ex. 58 MM D-Nva L-Asp L-Arg AcOH                                              Ex. 59 MM D-CHG L-Asp(Py) L-Arg AcOH                                          Ex. 60 EM D-CHG L-Asp L-Arg AcOH                                              CP 1 CH.sub.3 SO.sub.2 D-Leu -- L-Arg AcOH                                  ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        R.sub.2 --O(CH.sub.2 CH.sub.2 O).sub.n --CO--A.sub.1 --A.sub.2 -Gly-A.sub.    4 --pNA.HA                                                                      Product R.sub.2 n    A.sub.1 A.sub.2 A.sub.4                                                                             HA                               ______________________________________                                        Ex. 61                                                                              Me      1      D-Leu   --      L-Arg AcOH                                 Ex. 62 Me 2 D-Leu -- L-Arg AcOH                                               Ex. 63 Me 3 D-Leu -- L-Arg AcOH                                               Ex. 64 Me 7 D-Leu -- L-Arg AcOH                                               Ex. 65 Me 12 D-Leu -- L-Arg AcOH                                              Ex. 66 Me 16 D-Leu -- L-Arg AcOH                                              Ex. 67 Me 150 D-Leu -- L-Arg AcOH                                             Ex. 68 Me 161 D-Leu -- L-Arg AcOH                                             Ex. 69 Me 113 D-Leu -- L-Arg AcOH                                             Ex. 70 Me 45 D-Leu -- L-Arg AcOH                                              Ex. 71 Me 1 D-Nle -- L-Arg AcOH                                               Ex. 72 Me 2 D-Nle -- L-Arg AcOH                                               Ex. 73 Me 3 D-Nle -- L-Arg AcOH                                               Ex. 74 Me 45 D-Nle -- L-Arg AcOH                                              Ex. 75 Me 2 D-CHA -- L-Arg AcOH                                               Ex. 76 Me 7 D-CHA -- L-Arg AcOH                                               Ex. 77 Me 113 D-CHA -- L-Arg AcOH                                             Ex. 78 Me 2 D-CHT -- L-Arg AcOH                                               Ex. 79 Me 3 D-CHT -- L-Arg AcOH                                               Ex. 80 Me 7 D-Ala -- L-Arg AcOH                                               Ex. 81 Me 1 D-Leu L-Glu(Py) L-Arg HCl                                         Ex. 82 Me 7 D-Leu L-Glu(OMe) L-Arg HCl                                        Ex. 83 Me 16 D-Leu L-Glu(Pi) L-Arg HCl                                        Ex. 84 Et 2 D-Nle L-Asp(Py) L-Arg HCl                                         Ex. 85 Et 7 D-Nle L-Asp(OMe) L-Arg HCl                                        Ex. 86 Et 113 D-Nle L-Asp(Py) L-Lys HCl                                       Ex. 87 Et 2 D-CHA L-Glu(Py) L-Lys HCl                                         Ex. 88 Et 7 D-CHA L-Glu(OMe) L-Lys HCl                                        Ex. 89 Et 2 D-CHA L-Glu(Pi) L-Lys HCl                                         Ex. 90 Et 113 D-CHA L-Glu L-Lys HCl                                           Ex. 91 Et 2 D-Lys(Cbo) L-Glu L-Arg HCl                                        Ex. 92 Et 7 D-Lys(Cbo) L-Glu L-Arg HCl                                        Ex. 93 Et 2 D-Nva L-Glu L-Arg HCl                                             Ex. 94 Et 2 D-Nva L-Asp L-Arg HCl                                             Ex. 95 Et 7 D-Phe L-Glu L-Arg HCl                                             Ex. 96 Et 7 D-Phe L-Asp L-Arg HCl                                             Ex. 97 Et 7 D-Ile L-Glu L-Arg HCl                                             Ex. 98 Et 7 D-CHG L-Asp L-Arg HCl                                             Ex. 99 Bu 1 D-Leu -- L-Arg HCl                                                Ex. 100 Bu 7 D-Leu -- L-Arg HCl                                               Ex. 101 Bu 7 D-Nle -- L-Arg AcOH                                              Ex. 102 Bu 12 D-Nle -- L-Arg AcOH                                             Ex. 103 n-hexyl 1 D-Leu -- L-Arg AcOH                                         Ex. 104 n-hexyl 7 D-Leu -- L-Arg AcOH                                         Ex. 105 n-hexyl 1 D-CHA -- L-Arg AcOH                                         Ex. 106 n-hexyl 7 D-CHA -- L-Arg AcOH                                       ______________________________________                                    

The tri- and tetra-peptide compounds according to the invention can beprepared in accordance with a method known per se by the application ofconventional reaction mechanisms. The method of preparation which isrecommended here comprises reacting a peptide of the formula

    H-A.sub.1 -A.sub.2 -Gly-A.sub.4 -R                         (IV)

in which A₁, A₂, A₄ and R are defined as indicated above, with asubstance selected from the group consisting of

a) the oxymalonyl derivatives of the formula

    R.sub.1 --O--CO--CH.sub.2 --CO--T                          (V)

in which R₁ is defined as indicated above and T is OH, F, Cl or Br, and

b) the PEG derivatives of the formula

    R.sub.2 -O(CH.sub.2 CH.sub.2 O).sub.n -CO-Hal              (VI)

in which R₂ and n are as defined above and Hal is F, Cl or Br.

The reactions IV+V=I and IV+VI=I are each carried out in an inertsolvent such as DMF or THF, in the presence of an excess of a baseacting as a cosolvent and as a proton acceptor, such as Et₃ N, DIEA orany other suitable tertiary amine.

In practice, to carry out the reaction IV+V=I, two different techniquesare recommended according to the nature of the group T. When T is F, Clor Br, a molar ratio IV/V which is less than or equal to 1/2 will beused. When T is OH, the reaction will also be carried out in thepresence of a coupling agent such as Bop or HOBT (DCCI being added, ifappropriate, to the coupling agent HOBT where the latter is used), at atemperature of about 0° C. for at least 1 hour and then at RT for atleast 24 hours, with a molar ratio IV/V in the range from 1/1 to 1/1.5.

Furthermore, when T is F, Cl or Br in said reaction IV+V=I, a molarratio IV/Et₃ N of between 1.7/1 and 3.6/1 and preferably of 2/1 to 3.1/1will be used.

In practice, the reaction IV+VI=I will be carried out with a molar ratioIV/VI which is less than or equal to 1 and preferably less than or equalto 0.8.

To carry out the reaction IV+VI=I, it is preferred to use a derivativeof formula VI in which Hal is Cl. Such a chlorinated derivative, used asa starting material for said reaction IV+VI=I, can be synthesizedaccording to the following reaction scheme: ##STR7##

On the one hand the compounds of formula V are prepared from malonicacid and on the other hand the compounds of formula IV are obtained by aconventional method of peptide synthesis such as described especially indocument EP-A-0 280 610 cited above.

The compounds according to the invention are useful in the determinationof Factor Xa. An assay kit for the determination of Factor Xa, whichcontains at least one peptide compound selected from the groupconsisting of the compounds of formula I and their addition salts and,if appropriate, a standard sample of Factor Xa and of buffered dilutionmedia, is therefore also recommended.

A method of determining Factor Xa is also recommended, wherein a givenamount of a compound of formula I or of one of its addition salts isbrought into contact, in an aqueous biological medium, with a testsample (diluted if appropriate) which may contain Factor Xa.

Further advantages and characteristics of the invention will beunderstood more clearly from the following description of PreparatoryExamples and results of comparative tests. These data as a whole do notin any way imply a limitation but are given by way of illustration.

Preparation I

Preparation of MM-D-Leu-Gly-L-Arg-pNA.AcOH (Example 1)

a) Z-L-Arg-pNA.HCl

344 g (1 mol) of Z-L-Arg-OH.HCl are dissolved in anhydrous HMPT (freshlydistilled and dried over a molecular sieve) at RT and 139 ml (1 mol) ofEt₃ N are then added at RT, with stirring. 328 g (2 mol) ofp-nitrophenyl isocyanate are added to the resulting solution. Theresulting reaction medium is stirred for 24 h at RT and then evaporatedunder vacuum and the residue is taken up with the minimum amount of AcOHand then diluted with AcOEt. The resulting solution is subsequentlyextracted successively three times with small amounts of 0.5 M NaHCO₃,three times with a solution of KHSO₄ at 50 g/l and then several timeswith H₂ O semisaturated with NaCl. The organic phase is then dried overanhydrous sodium sulfate. After filtration (removal of Na₂ SO₄), thesolvent is evaporated off and the evaporation residue is recrystallizedfrom an AcOEt/MeOMe mixture (3/7 v/v) to give 350 g of the expectedproduct in the form of a white powder. M.p.=128-130° C.

Analysis (TLC on silica gel):

Rf=0.5 in AcOEt/pyridine/AcOH/H₂ O (20/4.5/3/1 v/v);

Rf=0.69 in CHCl₃ /MeOH/AcOH (5/3/1 v/v).

b) H-L-Arg-pNA.2HBr

100 g (0.215 mol) of Z-L-Arg-pNA.HCl are charged into a glass/Teflonapparatus. 800 ml of glacial AcOH, 200 ml of anisole and 1000 ml of asolution of HBr in glacial AcOH are added successively under an inertatmosphere (stream of nitrogen). The reaction is left to proceed for 1 hat RT under a nitrogen atmosphere. After this time has elapsed, thereaction mixture, which has become homogeneous during the deprotection,is precipitated in 20 l of ether (MeOMe or EtOEt). After decantation,the supernatant is discarded and the precipitate is washed several timeswith ether. The precipitate is collected by filtration and dried undervacuum over KOH for 24 h to give 94.2 g (yield: 96%) of the expectedproduct.

Analysis (TLC on silica gel):

Rf=0.04 in AcOEt/pyridine/AcOH/H₂ O (20/4.5/3/1.5 v/v);

Rf=0.38 in BuOH/AcOH/H₂ O (3/1/1 v/v).

c) Boc-Gly-L-Arg-DNA.HBr

1 g (2.19 mmol) of H-L-Arg-pNA.2HBr is dissolved in 10 ml of DMF, and0.854 ml (6.57 mmol) of DIEA is then added. In another vessel, asolution of 384 mg (2.19 mmol) of Boc-Gly-OH in 5 ml of DMF isneutralized with 0.285 ml of DIEA. The two solutions obtained in thisway are mixed and 970 mg of Bop are added to the resulting medium, thelatter being kept at RT; also, the pH is kept at a value of between 7and 8 by the addition of small portions of DIEA throughout the reaction.After one hour, the reaction has completely finished and the reactionmedium is evaporated to dryness under vacuum; the evaporation residue istaken up with an AcOEt/MeOH mixture and extracted with a 0.5 M aqueoussolution of NaHCO₃. The organic phase is dried over sodium sulfate,concentrated under vacuum and then precipitated in ether (MeOMe orEtOEt) to give 874 mg (yield: 75%) of the expected product.

Analysis (TLC on silica gel):

Rf=0.53 in CHCl₃ /MeOH/AcOH (10/3/1 v/v).

d) H-Gly-L-Arg-pNA.2H-TFA

874 mg (1.64 mmol) of Boc-Gly-L-Arg-pNA.HBr are charged into a reactorand 6.6 ml of CH₂ Cl₂ and 6.6 ml of H-TFA are then added successively.After a reaction time of 0.25 h at RT, the reaction mixture isprecipitated directly in ether. A flaky white precipitate is formedwhich is filtered off and dried to give 910 mg (yield: 96%) of theexpected product.

Analysis (TLC on silica gel):

Rf=0.09 in CHCl/MeOH/AcOH (5/3/1 v/v).

e) Boc-D-Leu-Gly-L-Arg-DNA.H-TFA

Following the procedures of Preparation Ic, a reaction mixturecomprising (i) 910 mg (1.57 mmol) of H-Gly-L-Arg-pNA.2H-TFA, (ii) 363 mg(1.57 mmol) of Boc-D-Leu-OH, (iii) 1.2 ml of DIEA and (iv) 695 mg (1.57mmol) of Bop is reacted in DMF and the reaction mixture is kept at RTfor 2 h, with stirring. After evaporation to dryness under vacuum, theevaporation residue is chromatographed on silica gel using a CHCl₃/MeOH/AcOH gradient (20/3/1 to 10/3/1 v/v) as the eluent. Thehomogeneous fractions collected are pooled and the eluent is evaporatedoff. Lyophilization gives 785 mg (yield: 80%) of the expected product inpure form.

Analysis (TLC on silica gel):

Rf=0.33 in CHCl₃ /MeOH/AcOH (10/3/1 v/v).

f) H-D-Leu-Gly-L-Ara-pNA.2H-TFA

785 mg (1.25 mmol) of Boc-D-Leu-Gly-L-Arg-pNA.H-TFA, obtained accordingto Preparation Ie, are reacted with 5 ml of CH₂ Cl₂ and 5 ml of H-TFA atRT. After 0.25 h, the reaction mixture is precipitated directly in etherto give a white precipitate, which is filtered off, washed with etherand dried. 779 mg (yield: 90%) of the expected product are collected.

Analysis (TLC on silica gel):

Rf=0.2 in CHCl₃ /MeOH/AcOH (5/3/1 v/v).

g) MM-D-Leu-Gly-L-Arg-pNA.AcOH

7.79 mg (1.12 mmol) of H-D-Leu-Gly-L-Arg-pNA.2H-TFA are charged into areaction vessel and 15 ml of DMF and 0.517 ml (3.705 mmnol) of Et₃ N arethen added. The resulting mixture is cooled to -30° C., with stirring,and 0.265 ml of methoxymalonyl chloride (MeO--CO--CH₂ --CO--Cl) is thenadded dropwise (still with stirring and at -30° C). The resultingmixture is kept at -30° C. for 0.5 h and then allowed to warm up to RT.After a reaction time of 2 h, the reaction mixture is filtered to removethe triethylammonium salt which has formed, and the filtrate isevaporated under vacuum. The evaporation residue is precipitated withether (EtoEt), filtered off and then chromatographed on an ion exchangeresin (AMBERLITE® IRA 401 S) acetylated beforehand, using an MeOH/H₂ Omixture (3/2 v/v) as the eluent. The homogeneous fractions are pooledand evaporated. Lyophilization of the resulting evaporation residuegives 566 mg (yield: 81%) of the expected product.

Analysis:

TLC on silica gel:

Rf=0.71 in CHCl₃ /MeOH/AcOH (10/3/1 v/v);

HPLC [on a column of HYPERSIL® C 18 (particle size: 3 μm) supplied byMERCK]:

TR=10 minutes [with an isocratic solution containing water (72.4%),acetonitrile (27.5%) and ACOH (0.2% w/w)].

Preparation II

Preparation of MM-D-Leu-L-Arg-pNA.AcOH (Example 1)

The procedure indicated in Preparation I is followed except that step Igis replaced with the following step:

1391 mg (2 mmol) of H-D-Leu-Gly-L-Arg-pNA.2H-TFA are charged into areaction vessel and 30 ml of DMF and 0.7 ml (5 mmol) of Et₃ N are thenadded. 973.5 mg of Bop are added at room temperature and a solution of259 mg of methoxymalonic acid (MeO--CO--CH₂ --CO--OH) in 10 ml of DMFand 0.7 ml of Et₃ N is then added to the resulting mixture. As thereaction proceeds, the pH is kept between 7 and 8 during the coupling bythe successive addition of small amounts of Et₃ N; after a reaction timeof 3 h, with stirring, the coupling is complete. The reaction medium isextracted with a solvent system of AcOEt/KHSO₄ (at 5% w/v) and thenAcOEt/0.5 M NaHCO₃. The AcOEt phase is then washed with watersemisaturated with NaCl. The AcOEt phase is evaporated under vacuum andthe evaporation residue is then chromatographed on a column of silica(particle size: 40-60 micrometers) using CHCl₃ /MeOH/AcOH (8/3/1 v/v) asthe eluent system. The homogeneous fractions are pooled and evaporated.Lyophilization of the resulting evaporation residue gives 812 mg (yield:65%) of the expected product.

Analysis:

TLC on silica gel:

Rf=0.71 in CHCl₃ /MeOH/AcOH (10/3/1 v/v);

HPLC [as indicated above in step Ig]:

TR=10 minutes.

Preparation III

Preparation of Me-OCH₂ CH₂ O-CO-D-Leu-Gly-L-Arg-pNA.AcOH (Example 61)

a) Et--OCH₂ CH₂ O--CO--Cl

Phosgene is condensed in a reactor at a temperature of -30° C. and 10 mlof ethylene glycol monomethyl ether, dissolved beforehand in THF, arethen added dropwise (still at -30° C.), by means of a dropping funnel,so that the phosgene is always in large excess relative to the alcohol.The reaction medium is stirred at -30° C. for 2 h and then allowed towarm up to RT. The excess phosgene is removed with a stream of nitrogen,the operation lasting several hours so that every trace of said phosgeneis removed. The reaction mixture is then evaporated under vacuum and theliquid obtained is used as such in the subsequent coupling step to give1320 mg (yield: 95%) of the expected product.

Analysis (IR spectrum):

disappearance of the alcohol band at 3300 cm⁻¹ ;

appearance of the chloroformate bands at 1776, 1152 and 690 cm⁻¹.

Comment: The method of step IIIa can be used directly to give the otherchloroformates of the formula R₂ --O(CH₂ CH₂ O)_(n) --CO--Cl.

b) MeOCH₂ CH₂ O-CO-D-Leu-Gly-L-Arg-pNA.AcOH

1391 mg (2 mmol) of H-D-Leu-Gly-L-Arg-pNA.2H-TFA (obtained as indicatedin Preparation If) are charged into a reaction vessel and 30 ml of DMFand 0.624 ml of Et₃ N are then added. The resulting mixture is cooled to0° C. by means of an ice bath. 0.304 g (2.2 mmol) of the chloroformateMeOCH₂ CH₂ O--CO--Cl is added to said mixture cooled in this way. Thereaction medium is stirred for 1 h and allowed to warm up to RT and thereaction is then continued for 1 h at RT. The reaction mixture is thenfiltered and evaporated under vacuum and the evaporation residue ischromatographed on an ion exchange resin (AMBERLITE® IRA 400 S)acetylated beforehand, using an MeOH/H₂ O mixture (3/2 v/v) as theeluent. The homogeneous fractions are pooled and evaporated.Lyophilization of the resulting evaporation residue gives 814 mg (yield:65%) of the expected product.

Analysis:

TLC on silica gel:

Rf=0.65 in CHCl₃ /MeOH/AcOH (10/3/1 v/v).

Preparation IV

Preparation of Me-O(CH₂ CH₂ O)₇ -CO-D-Leu-Gly-L-Arg-pNA.AcOH (Example64)

The procedure indicated in Preparation IIIb is followed except that theMe--OCH₂ CH₂ O--CO--Cl is replaced with MeO(CH₂ CH₂ O)₇ --COCl to give1.038 g (yield: 61%) of the expected product.

Analysis (TLC on silica gel):

Rf=0.61 in CHCl₃ /MeOH/AcOH (10/3/1 v/v);

Rf=0.14 in CHCl₃ /MeOH/AcOH (20/3/1 v/v).

Preparation V

Preparation of Me-O(CH₂ CH₂ O)₂ -CO-D-Leu-Gly-L-Arg-pNA.AcOH (Example62)

The procedure indicated in Preparation IIIb is followed except that theMe--OCH₂ CH₂ O--CO--Cl is replaced with MeO(CH₂ CH₂ O)₂ --COCl to give851 mg (yield: 68%) of the expected product.

Analysis (TLC on silica gel):

Rf=0.45 in CHCl₃ /MeOH/AcOH (15/3/1 v/v).

Comparative Tests

The activity of the peptides according to the invention towards FactorXa was determined by one of the conventional methods, such as the onedescribed in EP-A-0 280 160 (see page 14), the hydrolysis rate beingassessed by the variation in optical density with time (ΔOD/min). Theresults obtained at equimolar doses have been collated in Table IIIbelow, where the activity of the reference product CP 1 is specified asbeing equal to 100% for the sake of convenience.

The comparative results in Table III show that the peptides according tothe invention are at least as active as the reference product CP 1.

                  TABLE III                                                       ______________________________________                                        ACTIVITY TOWARDS FACTOR Xa                                                             Product  Activity                                                    ______________________________________                                               Ex. 1  115%                                                              Ex. 2  132%                                                                   Ex. 14 140%                                                                   Ex. 33 105%                                                                   Ex. 61 145%                                                                   Ex. 62 105%                                                                   Ex. 63 110%                                                                   Ex. 64 103%                                                                   Ex. 73 121%                                                                    Ex. 101 124%                                                                 CP 1 100%                                                                   ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= amino.sub.-- acid-1                             /note= - #"Leu, Ile, Nle(Norleucine), Nva(Norvaline),                         Phe, Ala, - # Lys, CHA(3-cyclohexylalanyl),                                   CHG(alpha-cy - #clohexylglycl) or                                             CHT(3-(4-hyd - #roxycyclohexyl)alanyl)"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #/label= amino.sub.-- acid-2                              /note= - #"Asp or Glu"                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/label= amino.sub.-- acid-4                              /note= - #"Arg or Lys"                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -      Xaa Xaa Gly Xaa                                                    __________________________________________________________________________

What is claimed is:
 1. A peptide compound selected from the groupconsisting of(a) MM-D-Leu-Gly-L-Arg-pNA, (b) EM-D-Leu-Gly-L-Arg-pNA, (c)MeO(CH₂ CH₂ O)-CO-D-Leu-Gly-L-Arg-pNA, (d) MeO(CH₂ CH₂ O)₃-D-Nle-Gly-L-Arg-pNA, (e) BuO(CH₂ CH₂ O)₇ -D-Nle-Gly-L-Arg-pNA, (f)MM-D-CRA-Gly-L-Arg-pNA, (g) MM-D-Nle-Gly-L-Arg-pNA, and (h) their acidaddition salts.
 2. A method of determining Factor Xa, wherein a givenamount of a peptide compound according to claim 1 or of one of itsaddition salts is brought into contact, in an aqueous biological medium,with a test sample which may contain Factor Xa.
 3. An assay kit for thedetermination of Factor Xa, which contains at least one peptide compoundselected from the group consisting of the compounds of formula I andtheir addition salts according to claim 1 and, if appropriate, astandard sample of Factor Xa and of buffered dilution media.